Search

Associating multiple sensory cues with objects and experience is a fundamental brain process that improves object recognition and memory performance. However, neural mechanisms that bind sensory features during learning and augment memory expression are unknown. Here we demonstrate multisensory appetitive and aversive memory in Drosophila. Combining colours and odours improved memory performance, even when each sensory modality was tested alone. Temporal control of neuronal function revealed visually selective mushroom body Kenyon cells (KCs) to be required for enhancement of both visual and olfactory memory after multisensory training. Voltage imaging in head-fixed flies showed that multisensory learning binds activity between streams of modality-specific KCs so that unimodal sensory input generates a multimodal neuronal response. Binding occurs between regions of the olfactory and visual KC axons, which receive valence-relevant dopaminergic reinforcement, and is propagated downstream. Dopamine locally releases GABAergic inhibition to permit specific microcircuits within KC-spanning serotonergic neurons to function as an excitatory bridge between the previously ‘modality-selective’ KC streams. Cross-modal binding thereby expands the KCs representing the memory engram for each modality into those representing the other. This broadening of the engram improves memory performance after multisensory learning and permits a single sensory feature to retrieve the memory of the multimodal experience.

Thirst increases astrocytic release and synthesis of D-serine to promote water procurement SCCMI uses single-cell sequencing to gain a whole-brain understanding of memory and motivational states. Study of thirst revealed the primary transcriptional response in the brain to occur in glial cells. Astrocytes upregulate the expression of an enzyme required to synthesise D-serine, a coagonist of neuronal NMDA-type glutamate receptors. D-serine, in turn, facilitates neural circuits that promote water procurement behaviours.

Associating multiple sensory cues with objects and experience is a fundamental brain process that improves object recognition and memory performance. However, neural mechanisms that bind sensory features during learning and augment memory expression are unknown. Here we demonstrate multisensory appetitive and aversive memory in Drosophila. Combining colors and odors improved memory performance, even when each sensory modality was tested alone. Temporal control of neuronal function revealed visually-selective mushroom body Kenyon Cells (KCs) to be required for both enhancement of visual and olfactory memory after multisensory training. Voltage imaging in head-fixed flies showed that multisensory learning binds activity between streams of modality-specific KCs, so that unimodal sensory input generates a multimodal neuronal response. Binding occurs between regions of the olfactory and visual KC axons, which receive valence-relevant dopaminergic reinforcement, and is propagated downstream. Dopamine locally releases GABA-ergic inhibition to permit specific microcircuits within KC-spanning serotonergic neurons to function as an excitatory bridge between the previously ‘modality-selective’ KC streams. Cross-modal binding thereby expands the olfactory memory engram by recruiting visual path KCs to become odor responsive. This broadening of the engram improves memory performance after multisensory learning and permits a single sensory feature to retrieve the memory of the multimodal experience.

Thirst emerges from a range of cellular changes that ultimately motivate an animal to consume water. Although thirst-responsive neuronal signals have been reported, the full complement of brain responses is unclear. Here we identify molecular and cellular adaptations in the brain using single-cell sequencing of water deprived Drosophila. Water deficiency primarily altered the glial transcriptome. Screening the regulated genes revealed astrocytic expression of the astray-encoded phosphoserine phosphatase to bi-directionally regulate water consumption. Astray synthesizes the gliotransmitter D-serine and vesicular release from astrocytes is required for drinking. Moreover, dietary D-serine rescues aay-dependent drinking deficits while facilitating water consumption and expression of water-seeking memory. D-serine action requires binding to neuronal NMDA-type glutamate receptors. Fly astrocytes contribute processes to tripartite synapses and the proportion of astrocytes that are themselves activated by glutamate increases with water deprivation. We propose that thirst elevates astrocytic D-serine release, which awakens quiescent glutamatergic circuits to enhance water procurement.

Cap-adjacent nucleotides of animal, protist and viral mRNAs can be O-methylated at the 2‘ position of the ribose (cOMe). The functions of cOMe in animals, however, remain largely unknown. Here we show that the two cap methyltransferases (CMTr1 and CMTr2) of Drosophila can methylate the ribose of the first nucleotide in mRNA. Double-mutant flies lack cOMe but are viable. Consistent with prominent neuronal expression, they have a reward learning defect that can be rescued by conditional expression in mushroom body neurons before training. Among CMTr targets are cell adhesion and signaling molecules. Many are relevant for learning, and are also targets of Fragile X Mental Retardation Protein (FMRP). Like FMRP, cOMe is required for localization of untranslated mRNAs to synapses and enhances binding of the cap binding complex in the nucleus. Hence, our study reveals a mechanism to co-transcriptionally prime mRNAs by cOMe for localized protein synthesis at synapses.

Animals use prior experience to assign absolute (good or bad) and also relative (better or worse) value to new experience. These learned values guide appropriate later decision-making. While our understanding of how the dopaminergic system computes absolute value is relatively advanced, the mechanistic underpinnings of relative valuation are unclear. Here we reveal mechanisms of absolute and relative aversive valuation in Drosophila. Three types of punishment-sensitive dopaminergic neurons (DANs) drive intensity-scaled plasticity at their respective mushroom body output neuron (MBON) connections to code absolute aversive value. In contrast, by comparing current and previous aversive experiences the MBON-DAN network can code relative aversive value by recruiting a specific subtype of reward-coding dopaminergic neurons which assigns a ‘better than’ value to the lesser of two aversive experiences. This study therefore provides an important functional consequence of having opposing populations of DANs and illustrates how these can operate together as a system within the MB network to code and compare sequential aversive experience to learn relative aversive value.

International audience; Animals use prior experience to assign absolute (good or bad) and relative (better or worse) value to new experience. These learned values guide appropriate later decision making. Even though our understanding of how the valuation system computes absolute value is relatively advanced, the mechanistic underpinnings of relative valuation are unclear. Here, we uncover mechanisms of absolute and relative aversive valuation in Drosophila. Three types of punishment-sensitive dopaminergic neurons (DANs) respond differently to electric shock intensity. During learning, these punishment-sensitive DANs drive intensity-scaled plasticity at their respective mushroom body output neuron (MBON) connections to code absolute aversive value. In contrast, by comparing the absolute value of current and previous aversive experiences, the MBON-DAN network can code relative aversive value by using specific punishment-sensitive DANs and recruiting a specific subtype of reward-coding DANs. Behavioral and physiological experiments revealed that a specific subtype of reward-coding DAN assigns a "better than" value to the lesser of the two aversive experiences. This study therefore highlights how appetitive-aversive system interactions within the MB network can code and compare sequential aversive experiences to learn relative aversive value.

Memory-relevant neuronal plasticity is believed to require local translation of new proteins at synapses. Understanding this process requires the visualization of the relevant mRNAs within these neuronal compartments. Here, we used single-molecule fluorescence in situ hybridization to localize mRNAs at subcellular resolution in the adult Drosophila brain. mRNAs for subunits of nicotinic acetylcholine receptors and kinases could be detected within the dendrites of co-labeled mushroom body output neurons (MBONs) and their relative abundance showed cell specificity. Moreover, aversive olfactory learning produced a transient increase in the level of CaMKII mRNA within the dendritic compartments of the γ5β'2a MBONs. Localization of specific mRNAs in MBONs before and after learning represents a critical step towards deciphering the role of dendritic translation in the neuronal plasticity underlying behavioral change in Drosophila.

We present here our reflections on the scientific work of the late Troy D. Zars (1967 – 2018), on what it was like to work with him, and what it means to us. A common theme running through his work is that memory systems are not for replaying the past. Rather, they are forward-looking systems, providing whatever guidance past experience has to offer for anticipating the outcome of future actions. And in situations where no such guidance is available trying things out is the best option. Working with Troy was inspiring precisely because of the optimism inherent in this concept and that he himself embodied. Our reflections highlight what this means to us as his former mentors, colleagues, and mentees, respectively, and what it might mean for the future of neurogenetics.

Summary Prior experience of a stimulus can inhibit subsequent acquisition or expression of a learned association of that stimulus. However, the neuronal manifestations of this learning effect, named latent inhibition (LI), are poorly understood. Here, we show that prior odor exposure can produce context-dependent LI of later appetitive olfactory memory performance in Drosophila. Odor pre-exposure forms a short-lived aversive memory whose lone expression lacks context-dependence. Acquisition of odor pre-exposure memory requires aversively reinforcing dopaminergic neurons that innervate two mushroom body compartments—one group of which exhibits increasing activity with successive odor experience. Odor-specific responses of the corresponding mushroom body output neurons are suppressed, and their output is necessary for expression of both pre-exposure memory and LI of appetitive memory. Therefore, odor pre-exposure attaches negative valence to the odor itself, and LI of appetitive memory results from a temporary and context-dependent retrieval deficit imposed by competition with the parallel short-lived aversive memory., Highlights • Odor pre-exposure alters the expression of a learned association of that odor • Pre-exposure memory only affects subsequent retrieval if context is consistent • Pre-exposure memory can complement or compete with a learned association • Odor pre-exposure forms a labile mushroom body-dependent aversive memory, Jacob et al. show that prior experience of an odor can inhibit the retrieval of a newly formed memory for that same odor. Odor pre-exposure forms a labile mushroom body-dependent aversive memory that competes with expression of new learning if the context is appropriate. This study explains how a retrieval defect can underlie latent inhibition.

Prior experience of a stimulus can inhibit subsequent acquisition or expression of a learned association of that stimulus. However, the neuronal manifestations of this learning effect, named latent inhibition (LI), are poorly understood. Here we show that odor pre-exposure produces LI of appetitive olfactory memory performance in Drosophila. Behavioral expression of LI requires that the context during memory testing resembles that during the odor pre-exposures. Odor pre-exposure forms an aversive memory that requires dopaminergic neurons that innervate the γ2α′1 and α3 mushroom body compartments - those to α3 exhibit increasing odor-driven activity with successive pre-exposures. In contrast, odor-specific responses of the corresponding mushroom body output neurons are suppressed. Odor pre-exposure therefore recruits specific dopaminergic neurons that provide teaching signals that attach negative valence to the odor itself. LI of Drosophila appetitive memory consequently results from a temporary and context-dependent retrieval deficit imposed by competition with this short-lived aversive memory.

Making inferences about the computations performed by neuronal circuits from synapse-level connectivity maps is an emerging opportunity in neuroscience. The mushroom body (MB) is well positioned for developing and testing such an approach due to its conserved neuronal architecture, recently completed dense connectome, and extensive prior experimental studies of its roles in learning, memory, and activity regulation. Here, we identify new components of the MB circuit inDrosophila, including extensive visual input and MB output neurons (MBONs) with direct connections to descending neurons. We find unexpected structure in sensory inputs, in the transfer of information about different sensory modalities to MBONs, and in the modulation of that transfer by dopaminergic neurons (DANs). We provide insights into the circuitry used to integrate MB outputs, connectivity between the MB and the central complex and inputs to DANs, including feedback from MBONs. Our results provide a foundation for further theoretical and experimental work.

Dietary magnesium (Mg2+) supplementation can enhance memory in young and aged rats. Memory-enhancing capacity was largely ascribed to increases in hippocampal synaptic density and elevated expression of the NR2B subunit of the NMDA-type glutamate receptor. Here we show that Mg2+ feeding also enhances long-term memory in Drosophila. Normal and Mg2+-enhanced fly memory appears independent of NMDA receptors in the mushroom body and instead requires expression of a conserved CNNM-type Mg2+-efflux transporter encoded by the unextended (uex) gene. UEX contains a putative cyclic nucleotide-binding homology domain and its mutation separates a vital role for uex from a function in memory. Moreover, UEX localization in mushroom body Kenyon cells (KCs) is altered in memory-defective flies harboring mutations in cAMP-related genes. Functional imaging suggests that UEX-dependent efflux is required for slow rhythmic maintenance of KC Mg2+. We propose that regulated neuronal Mg2+ efflux is critical for normal and Mg2+-enhanced memory., eLife digest The proverbial saying ‘you are what you eat’ perfectly summarizes the concept that our diet can influence both our mental and physical health. We know that foods that are good for the heart, such as nuts, oily fish and berries, are also good for the brain. We know too that vitamins and minerals are essential for overall good health. But is there any evidence that increasing your intake of specific vitamins or minerals could help boost your brain power? While it might sound almost too good to be true, there is some evidence that this is the case for at least one mineral, magnesium. Studies in rodents have shown that adding magnesium supplements to food improves how well the animals perform on memory tasks. Both young and old animals benefit from additional magnesium. Even elderly rodents with a condition similar to Alzheimer’s disease show less memory loss when given magnesium supplements. But what about other species? Wu et al. now show that magnesium supplements also boost memory performance in fruit flies. One group of flies was fed with standard cornmeal for several days, while the other group received cornmeal supplemented with magnesium. Both groups were then trained to associate an odor with a food reward. Flies that had received the extra magnesium showed better memory for the odor when tested 24 hours after training. Wu et al. show that magnesium improves memory in the flies via a different mechanism to that reported previously for rodents. In rodents, magnesium increased levels of a receptor protein for a brain chemical called glutamate. In fruit flies, by contrast, the memory boost depended on a protein that transports magnesium out of neurons. Mutant flies that lacked this transporter showed memory impairments. Unlike normal flies, those without the transporter showed no memory improvement after eating magnesium-enriched food. The results suggest that the transporter may help adjust magnesium levels inside brain cells in response to neural activity. Humans produce four variants of this magnesium transporter, each encoded by a different gene. One of these transporters has already been implicated in brain development. The findings of Wu et al. suggest that the transporters may also act in the adult brain to influence cognition. Further studies are needed to test whether targeting the magnesium transporter could ultimately hold promise for treating memory impairments.

Memory-relevant neuronal plasticity is believed to require local translation of new proteins at synapses. Understanding this process requires the visualization of the relevant mRNAs within these neuronal compartments. Here we used single-molecule fluorescence in situ hybridization (smFISH) to localize mRNAs at subcellular resolution in the adult Drosophila brain. mRNAs for subunits of nicotinic acetylcholine receptors and kinases could be detected within the dendrites of co-labelled Mushroom Body Output Neurons (MBONs) and their relative abundance showed cell-specificity. Moreover, aversive olfactory learning produced a transient increase in the level of CaMKII mRNA within the dendritic compartments of the γ5β′2a MBONs. Localization of specific mRNAs in MBONs before and after learning represents a critical step towards deciphering the role of dendritic translation in the neuronal plasticity underlying behavioural change in Drosophila.

Making inferences about the computations performed by neuronal circuits from synapse-level connectivity maps is an emerging opportunity in neuroscience. The mushroom body (MB) is well positioned for developing and testing such an approach due to its conserved neuronal architecture, recently completed dense connectome, and extensive prior experimental studies of its roles in learning, memory and activity regulation. Here we identify new components of the MB circuit inDrosophila, including extensive visual input and MB output neurons (MBONs) with direct connections to descending neurons. We find unexpected structure in sensory inputs, in the transfer of information about different sensory modalities to MBONs, and in the modulation of that transfer by dopaminergic neurons (DANs). We provide insights into the circuitry used to integrate MB outputs, connectivity between the MB and the central complex and inputs to DANs, including feedback from MBONs. Our results provide a foundation for further theoretical and experimental work.

The gut microbiome has been proposed to influence diverse behavioral traits of animals, although the experimental evidence is limited and often contradictory. Here, we made use of the tractability of Drosophila melanogaster for both behavioral analyses and microbiome studies to test how elimination of microorganisms affects a number of behavioral traits. Relative to conventional flies (i.e. with unaltered microbiome), microbiologically sterile (axenic) flies displayed a moderate reduction in memory performance in olfactory appetitive conditioning and courtship assays. The microbiological status of the flies had a small or no effect on anxiety-like behavior (centrophobism) or circadian rhythmicity of locomotor activity, but axenic flies tended to sleep for longer and displayed reduced sleep rebound after sleep deprivation. These last two effects were robust for most tests conducted on both wild-type Canton S and w1118 strains, as well for tests using an isogenized panel of flies with mutations in the period gene, which causes altered circadian rhythmicity. Interestingly, the effect of absence of microbiota on a few behavioral features, most notably instantaneous locomotor activity speed, varied among wild-type strains. Taken together, our findings demonstrate that the microbiome can have subtle but significant effects on specific aspects of Drosophila behavior, some of which are dependent on genetic background., Highlighted Article: Drosophila experimentally deprived of their gut microbiome display changes in learning and memory, and in the duration of sleep under standard conditions and following sleep deprivation.

This special issue is dedicated to ‘Future Perspectives of Neurogenetics’ in honor of Troy D. Zars (1967–2018). It is intended to offer insight and inspiration for our understanding of how adaptive...

Summary Different types of Drosophila dopaminergic neurons (DANs) reinforce memories of unique valence and provide state-dependent motivational control [1]. Prior studies suggest that the compartment architecture of the mushroom body (MB) is the relevant resolution for distinct DAN functions [2, 3]. Here we used a recent electron microscope volume of the fly brain [4] to reconstruct the fine anatomy of individual DANs within three MB compartments. We find the 20 DANs of the γ5 compartment, at least some of which provide reward teaching signals, can be clustered into 5 anatomical subtypes that innervate different regions within γ5. Reconstructing 821 upstream neurons reveals input selectivity, supporting the functional relevance of DAN sub-classification. Only one PAM-γ5 DAN subtype γ5(fb) receives direct recurrent feedback from γ5β′2a mushroom body output neurons (MBONs) and behavioral experiments distinguish a role for these DANs in memory revaluation from those reinforcing sugar memory. Other DAN subtypes receive major, and potentially reinforcing, inputs from putative gustatory interneurons or lateral horn neurons, which can also relay indirect feedback from MBONs. We similarly reconstructed the single aversively reinforcing PPL1-γ1pedc DAN. The γ1pedc DAN inputs mostly differ from those of γ5 DANs and they cluster onto distinct dendritic branches, presumably separating its established roles in aversive reinforcement and appetitive motivation [5, 6]. Tracing also identified neurons that provide broad input to γ5, β′2a, and γ1pedc DANs, suggesting that distributed DAN populations can be coordinately regulated. These connectomic and behavioral analyses therefore reveal further complexity of dopaminergic reinforcement circuits between and within MB compartments., Highlights • Nanoscale anatomy reveals additional subtypes of rewarding dopaminergic neurons • Connectomics reveals input specificity to subtypes of dopaminergic neurons • Axon morphology implies dopaminergic neurons provide subcompartment-level function • Unique dopaminergic subtypes serve aversive memory extinction and sugar learning, Otto et al. use electron microscope resolution connectomics to describe the structure and organization of neurons providing synaptic input to functionally discrete subtypes of dopaminergic neurons. The nanoscale anatomy reveals further anatomical and functional specialization of dopaminergic neurons, which is confirmed with behavioral experiments.

Foraging strategies emerge from genetically encoded programs that are similar across animal species. Here we examine circuits that control a conserved foraging state, local search behavior after food removal, in Caenorhabditis elegans. We show that local search is triggered by two parallel groups of chemosensory and mechanosensory glutamatergic neurons that detect food-related cues. Each group of sensory neurons suppresses distinct integrating neurons through a G protein-coupled metabotropic glutamate receptor, MGL-1, to release local search. The chemosensory and mechanosensory modules are separate and redundant; glutamate release from either module can drive the full behavior. A transition from local search to global search over several minutes after food removal is associated with two changes in circuit function. First, the spontaneous activity of sensory neurons falls. Second, the motor pattern generator for local search becomes less responsive to sensory input. This multimodal, distributed short-term food memory provides robust control of an innate behavior.

Neurostimulation techniques allow us to manipulate the activity of nervous systems, and even that of single neurons. In this piece, researchers discuss what they see as the current key bottlenecks to controlling neural biology.

The gut microbiome has been proposed to influence diverse behavioral traits of animals, although the experimental evidence is limited and often contradictory. Here, we make use of the tractability of Drosophila melanogaster for both behavioral analyses and microbiome studies to test how elimination of microorganisms affects a number of behavioral traits. Relative to conventional flies (i.e., with unaltered microbiome), microbiologically-sterile (axenic) flies displayed a moderate reduction in memory performance in olfactory appetitive conditioning and courtship assays. The microbiological status of the flies had small or no effect on anxiety-like behavior (centrophobism) or circadian rhythmicity of locomotor activity, but axenic flies tended to sleep for longer and displayed reduced sleep rebound after sleep deprivation. The latter effects were robust for most tests conducted on both wildtype Canton S and w1118 strains, as well for tests using an isogenized panel of flies with mutations in the period gene, which causes altered circadian rhythmicity. Interestingly, the effect of absence of microbiota on a few behavioral features, most notably instantaneous locomotor activity speed, varied among wild-type strains. Taken together, our findings demonstrate that the microbiome can have subtle but significant effects on specific aspects of Drosophila behavior, some of which are dependent on genetic background.

TheDrosophilaventral nerve cord (VNC) receives and processes descending signals from the brain to produce a variety of coordinated locomotor outputs. It also integrates sensory information from the periphery and sends ascending signals to the brain. We used single-cell transcriptomics to generate an unbiased classification of cellular diversity in the VNC of five-day old adult flies. We produced an atlas of 26,000 high-quality cells, representing more than 100 transcriptionally distinct cell types. The predominant gene signatures defining neuronal cell types reflect shared developmental histories based on the neuroblast from which cells were derived, as well as their birth order. Cells could also be assigned to specific neuromeres using adult Hox gene expression. This single-cell transcriptional atlas of the adult fly VNC will be a valuable resource for future studies of neurodevelopment and behavior.

Somatic transposon expression in neural tissue is commonly considered as a measure of mobilization and has therefore been linked to neuropathology and organismal individuality. We combined genome sequencing data with single-cell mRNA sequencing of the same inbred fly strain to map transposon expression in the Drosophila midbrain and found that transposon expression patterns are highly stereotyped. Every detected transposon is resident in at least one cellular gene with a matching expression pattern. Bulk RNA sequencing from fly heads of the same strain revealed that coexpression is a physical link in the form of abundant chimeric transposon–gene mRNAs. We identified 264 genes where transposons introduce cryptic splice sites into the nascent transcript and thereby significantly expand the neural transcript repertoire. Some genes exclusively produce chimeric mRNAs with transposon sequence; on average, 11.6% of the mRNAs produced from a given gene are chimeric. Conversely, most transposon-containing transcripts are chimeric, which suggests that somatic expression of these transposons is largely driven by cellular genes. We propose that chimeric mRNAs produced by alternative splicing into polymorphic transposons, rather than transposon mobilization, may contribute to functional differences between individual cells and animals.

Forming long-term memory (LTM) in many cases requires repetitive experience spread over time. InDrosophila, aversive olfactory LTM is optimal following spaced training, multiple trials of differential odor conditioning with rest intervals. Studies often compare memory after spaced to that after massed training, same number of trials without interval. Here we show flies acquire additional information after spaced training, forming an aversive memory for the shock-paired odor and a ‘safety-memory’ for the explicitly unpaired odor. Safety-memory requires repetition, order and spacing of the training trials and relies on specific subsets of rewarding dopaminergic neurons. Co-existence of the aversive and safety memories can be measured as depression of odor-specific responses at different combinations of junctions in the mushroom body output network. Combining two particular outputs appears to signal relative safety. Learning a complementary safety memory thereby augments LTM performance after spaced training by making the odor preference more certain.

Summary Memories are stored in the fan-out fan-in neural architectures of the mammalian cerebellum and hippocampus and the insect mushroom bodies. However, whereas key plasticity occurs at glutamatergic synapses in mammals, the neurochemistry of the memory-storing mushroom body Kenyon cell output synapses is unknown. Here we demonstrate a role for acetylcholine (ACh) in Drosophila. Kenyon cells express the ACh-processing proteins ChAT and VAChT, and reducing their expression impairs learned olfactory-driven behavior. Local ACh application, or direct Kenyon cell activation, evokes activity in mushroom body output neurons (MBONs). MBON activation depends on VAChT expression in Kenyon cells and is blocked by ACh receptor antagonism. Furthermore, reducing nicotinic ACh receptor subunit expression in MBONs compromises odor-evoked activation and redirects odor-driven behavior. Lastly, peptidergic corelease enhances ACh-evoked responses in MBONs, suggesting an interaction between the fast- and slow-acting transmitters. Therefore, olfactory memories in Drosophila are likely stored as plasticity of cholinergic synapses., Highlights • Mushroom body Kenyon cell function requires ChAT and VAChT expression • Kenyon cell-released acetylcholine drives mushroom body output neurons • Blocking nicotinic receptors impairs mushroom body output neuron activation • Acetylcholine interacts with coreleased neuropeptide, Fruit fly memory involves plasticity of mushroom body synapses. Barnstedt et al. identified acetylcholine as the mushroom body neurotransmitter. Mushroom body output neuron activation requires nicotinic acetylcholine receptors. Impaired receptor function reduces physiological responses and alters odor-driven behavior.

Two recent studies in Drosophila provide evidence that dopamine can drive synaptic depression and facilitation, supporting models in which learning and the behavioral state of the fly guide behavior by tuning mushroom body output synapses.

Summary Animals must express the appropriate behavior that meets their most pressing physiological needs and their environmental context. However, it is currently unclear how alternative behavioral options are evaluated and appropriate actions are prioritized. Here, we describe how fruit flies choose between feeding and courtship; two behaviors necessary for survival and reproduction. We show that sex- and food-deprived male flies prioritize feeding over courtship initiation, and manipulation of food quality or the animal’s internal state fine-tunes this decision. We identify the tyramine signaling pathway as an essential mediator of this decision. Tyramine biosynthesis is regulated by the fly’s nutritional state and acts as a satiety signal, favoring courtship over feeding. Tyramine inhibits a subset of feeding-promoting tyramine receptor (TyrR)-expressing neurons and activates P1 neurons, a known command center for courtship. Conversely, the perception of a nutritious food source activates TyrR neurons and inhibits P1 neurons. Therefore, TyrR and P1 neurons are oppositely modulated by starvation, via tyramine levels, and food availability. We propose that antagonistic co-regulation of neurons controlling alternative actions is key to prioritizing competing drives in a context- dependent manner., Graphical abstract, Highlights • Drosophila males deprived of both food and sex have competing needs • Choosing between feeding or mating is modulated by food quality and internal drive • Tyramine signaling mediates the choice between feeding and courtship • Antagonism between feeding and courtship neurons determines the behavior selected, How are alternative options evaluated in the brain and specific actions prioritized? Cheriyamkunnel et al. describe a novel neural mechanism by which fruit flies balance and prioritize the competing needs of feeding and mating by integrating nutrient availability and motivational drives.

Summary Nervous systems contain sensory neurons, local neurons, projection neurons, and motor neurons. To understand how these building blocks form whole circuits, we must distil these broad classes into neuronal cell types and describe their network connectivity. Using an electron micrograph dataset for an entire Drosophila melanogaster brain, we reconstruct the first complete inventory of olfactory projections connecting the antennal lobe, the insect analog of the mammalian olfactory bulb, to higher-order brain regions in an adult animal brain. We then connect this inventory to extant data in the literature, providing synaptic-resolution “holotypes” both for heavily investigated and previously unknown cell types. Projection neurons are approximately twice as numerous as reported by light level studies; cell types are stereotyped, but not identical, in cell and synapse numbers between brain hemispheres. The lateral horn, the insect analog of the mammalian cortical amygdala, is the main target for this olfactory information and has been shown to guide innate behavior. Here, we find new connectivity motifs, including axo-axonic connectivity between projection neurons, feedback, and lateral inhibition of these axons by a large population of neurons, and the convergence of different inputs, including non-olfactory inputs and memory-related feedback onto third-order olfactory neurons. These features are less prominent in the mushroom body calyx, the insect analog of the mammalian piriform cortex and a center for associative memory. Our work provides a complete neuroanatomical platform for future studies of the adult Drosophila olfactory system., Graphical Abstract, Highlights • First complete parts list for second-order neurons of an adult olfactory system • Quantification of left-right stereotypy in cell and synapse number • Axo-axonic connections form hierarchical communities in the lateral horn • Local and memory-related feedback target projection neuron axons, Bates, Schlegel et al. report the first complete part list of second-order neurons in an adult arthropod olfactory system. They show that these neurons are stereotyped across hemispheres and reveal their hierarchical interconnectivity and how they connect to third-order targets and memory-related neurons.

Summary Forming long-term memory (LTM) often requires repetitive experience spread over time. Studies in Drosophila suggest aversive olfactory LTM is optimal after spaced training, multiple trials of differential odor conditioning with rest intervals. Memory after spaced training is frequently compared to that after the same number of trials without intervals. Here we show that, after spaced training, flies acquire additional information and form an aversive memory for the shock-paired odor and a slowly emerging and more persistent “safety-memory” for the explicitly unpaired odor. Safety-memory acquisition requires repetition, order, and spacing of the training trials and relies on triggering specific rewarding dopaminergic neurons. Co-existence of aversive and safety memories is evident as depression of odor-specific responses at different combinations of junctions in the mushroom body output network; combining two outputs appears to signal relative safety. Having complementary aversive and safety memories augments LTM performance after spaced training by making the odor preference more certain., Highlights • Spaced training forms complementary long-term aversive and safety memories • Safety memory is protein-synthesis-dependent long-term memory • Safety memory acquisition requires repetition, order, and spacing of trials • Specific dopaminergic neurons reinforce the delayed recognition of safety, Multiple trials of aversive olfactory conditioning in Drosophila can form an aversive memory for one odor and an unexpected safety memory for the other. Although safety memory appears slowly after training, it lasts longer than aversive memory.

Accurately predicting an outcome requires that animals learn supporting and conflicting evidence from sequential experience. In mammals and invertebrates, learned fear responses can be suppressed by experiencing predictive cues without punishment, a process called memory extinction. Here, we show that extinction of aversive memories in Drosophila requires specific dopaminergic neurons, which indicate that omission of punishment is remembered as a positive experience. Functional imaging revealed co-existence of intracellular calcium traces in different places in the mushroom body output neuron network for both the original aversive memory and a new appetitive extinction memory. Light and ultrastructural anatomy are consistent with parallel competing memories being combined within mushroom body output neurons that direct avoidance. Indeed, extinction-evoked plasticity in a pair of these neurons neutralizes the potentiated odor response imposed in the network by aversive learning. Therefore, flies track the accuracy of learned expectations by accumulating and integrating memories of conflicting events.

Highlights • Dopamine neurons coding opposite value are segregated in the fly brain. • Dopamine neurons representing reward type innervate discrete mushroom body areas. • Mushroom body output neurons are specific to dopaminergic reinforcement zones. • Olfactory learning skews different collections of mushroom body output junctions. • Mushroom bodies are fan-out fan-in circuits like the cerebellum and hippocampus., Learning permits animals to attach meaning and context to sensory stimuli. How this information is coded in neural networks in the brain, and appropriately retrieved and utilized to guide behavior, is poorly understood. In the fruit fly olfactory memories of particular value are represented within sparse populations of odor-activated Kenyon cells (KCs) in the mushroom body ensemble. During learning reinforcing dopaminergic neurons skew the mushroom body network by driving zonally restricted plasticity at synaptic junctions between the KCs and subsets of the overall small collection of mushroom body output neurons. Reactivation of this skewed KC-output neuron network retrieves memory of odor valence and guides appropriate approach or avoidance behavior.

To understand the brain, molecular details need to be overlaid onto neural wiring diagrams so that synaptic mode, neuromodulation and critical signaling operations can be considered. Single-cell transcriptomics provide a unique opportunity to collect this information. Here we present an initial analysis of thousands of individual cells from Drosophila midbrain, that were acquired using Drop-Seq. A number of approaches permitted the assignment of transcriptional profiles to several major brain regions and cell-types. Expression of biosynthetic enzymes and reuptake mechanisms allows all the neurons to be typed according to the neurotransmitter or neuromodulator that they produce and presumably release. Some neuropeptides are preferentially co-expressed in neurons using a particular fast-acting transmitter, or monoamine. Neuromodulatory and neurotransmitter receptor subunit expression illustrates the potential of these molecules in generating complexity in neural circuit function. This cell atlas dataset provides an important resource to link molecular operations to brain regions and complex neural processes.

Highlights • Simple and rapid smFISH protocol suitable for medium throughput. • Sensitive mRNA detection deep in whole-mount larval and adult Drosophila brains. • Multiplexed detection of RNA in combination with antibody staining. • Quantitation of primary transcription and post-transcriptional mRNA levels. • Reliable cell type markers in a whole-mount brain complementary to antibody markers., RNA in situ hybridization is a powerful method to investigate post-transcriptional regulation, but analysis of intracellular mRNA distributions in thick, complex tissues like the brain poses significant challenges. Here, we describe the application of single-molecule fluorescent in situ hybridization (smFISH) to quantitate primary nascent transcription and post-transcriptional regulation in whole-mount Drosophila larval and adult brains. Combining immunofluorescence and smFISH probes for different regions of a single gene, i.e., exons, 3′UTR, and introns, we show examples of a gene that is regulated post-transcriptionally and one that is regulated at the level of transcription. Our simple and rapid protocol can be used to co-visualise a variety of different transcripts and proteins in neuronal stem cells as well as deep brain structures such as mushroom body neuropils, using conventional confocal microscopy. Finally, we introduce the use of smFISH as a sensitive alternative to immunofluorescence for labelling specific neural stem cell populations in the brain.

The ability to record neural activity in the brain of a living organism at cellular resolution is of great importance for defining the neural circuit mechanisms that direct behavior. Here we present an adaptive two-photon microscope optimized for extraction of neural signals over volumes in intact Drosophila brains, even in the presence of specimen motion. High speed volume imaging was made possible through reduction of spatial resolution while maintaining the light collection efficiency of a high resolution, high numerical aperture microscope. This enabled simultaneous recording of odor-evoked calcium transients in a defined volume of mushroom body Kenyon cell bodies in a live fruit fly.

Animals constantly reassess the reliability of learned information to optimize their behavior. On retrieval, consolidated long-term memory can be neutralized by extinction if the learned prediction was inaccurate 1. Alternatively, retrieved memory can be maintained, following a period of reconsolidation during which it is labile 2. Although extinction and reconsolidation provide opportunities to alleviate problematic human memories 3–5, we lack a detailed mechanistic understanding of memory updating processes. Here we identify neural operations underpinning re-evaluation of memory in Drosophila. Reactivation of sugar-reinforced olfactory memory can lead to either extinction or reconsolidation, depending on prediction accuracy. Each process recruits activity in specific parts of the mushroom body output network and distinct subsets of reinforcing dopaminergic neurons. Memory extinction requires output neurons with dendrites in the α and α′ lobes of the mushroom body, which drive negatively reinforcing dopaminergic neurons that innervate neighbouring zones. The aversive valence of these new extinction memories neutralizes previously learned odor preference. Memory reconsolidation requires the γ2α′ 1 mushroom body output neurons. This pathway recruits negatively reinforcing dopaminergic neurons innervating the same compartment and re-engages positively reinforcing dopaminergic neurons to reconsolidate the original reward memory. These data establish that recurrent and hierarchical connectivity between mushroom body output neurons and dopaminergic neurons enables memory re-evaluation driven by reward prediction error.

Drinking water is innately rewarding to thirsty animals. In addition, the consumed value can be assigned to behavioral actions and predictive sensory cues by associative learning. Here we show that thirst converts water avoidance into water-seeking in naive Drosophila melanogaster. Thirst also permitted flies to learn olfactory cues paired with water reward. Water learning required water taste and